Testing Thrombin Sepharose Beads and Glutathione Resin
Date: 08/30/21
GST tagged protein from CDI (received in March 2018, pick 2 proteins for this test experiment) • STAT3: 3.1mg/ml
• (also GST protein from Sigma cat# G5663, ~1mg/ml control protein for GST tag)
Dilute protein samples to 0.1ug/ul or 1ug/ul (in 1x PBS, pH7.4) based on their concentrations given by CDI
Thrombin cleavage
Thrombin Sepharose Beads (BioVision Cat. # 7925-1)
The target fusion protein should be purified to homogeneity and dialyzed against 50 mM Tris buffer, 0.1 M NaCl, pH 8.0 before setting up the cleavage reaction.
Take 20ul of sample (2 or 20ug of GST-tagged protein); add 1ul of 1M Tris, pH8 (final 50 mM Tris); make 3x 10ul aliquots (for 0, 1 and 2h time points)
1) STAT3_1ug_0
2) STAT3_1ug_1h
3) STAT3_1ug_2h
4) STAT3_10ug_0
5) STAT3_10ug_1h
6) STAT3_10ug_2h
Resuspend the beads by gentle swirling. Do not Vortex.
Aliquot 2.5μl of the suspended slurry and add to 1h or 2h digestion tubes containing the fusion protein
• Recommended resin: protein ratio is 15ul slurry to 1mg of fusion protein
Mix gently by inverting the tube (do not vortex) and gently shake on a rotary shaker at room temperature.
• Recommended concentration of target fusion protein is 1 mg/ml, but we don’t have that much protein and concentration is low in our samples.
At each time point, take the corresponding tube, spin down the reaction for 2-3 m @ 5000rpm
Transfer the supernatant to a fresh tube (“Reaction”)
Wash the resin with 10ul of 1xPBS, spin down the reaction for 2-3 m @ 5000rpm
Transfer the supernatant to a fresh tube (“Wash”)
Take either 50% or 10% (see below for loading) from each tube to be analyzed on gel
Add LDS and DTT to each sample (final 1x LDS, 50mM DTT)
Heat samples at 70C for 10’
Run 4-12% Bis-Tris gel
1) Marker
2) STAT3_1ug_0 (load 500ng)
3) STAT3_1ug_1h_reaction (load 50%, 500ng)
4) STAT3_1ug_1h_wash (50%)
5) STAT3_1ug_2h_reaction (50%)
6) STAT3_1ug_2h_wash (50%)
7) STAT3_10ug_0
8) STAT3_10ug_1h_reaction (load 10%, 1ug)
9) STAT3_10ug_1h_wash (10%)
10) STAT3_10ug_2h_reaction (10%)
11) STAT3_10ug_2h_wash (10%)
12) GST_100ng (10%)
13) GST_250ng
14) GST_500ng
15) BSA_100ng
The Thrombin Sepharose beads worked very poorly.
Check the formulation of thrombin beads: 6% cross linked Sepharose beads provided as 50% slurry in pure glycerol
The reaction in the above experiment has 10ul of protein and 2.5ul of resin, the final glycerol concentration is 20%. That might affect the digestion efficiency.
Thrombin cleavage and removal of GST by Glutathione resin
Get another GST fusion protein from Peter (GST-PP7 @ 3.5mg/ml)
Dilute STAT3 (3.1mg/ml) and GST-PP7 to 0.1mg/ml with 50mM Tris, pH8, 0.1M NaCl
Set up the following reaction:
100ul of protein @ 0.1ug/ul (5ug per reaction); add 10ul of thrombin resin
Mix resin with protein by pipetting
Put the tubes thermomixer set @ 25C, 600rpm
Time course: 0, 2, 4, 20hr
At each time point, take the corresponding tube, spin down the reaction for 2-3 m @ 5000rpm
Transfer 10ul of the supernatant from each tube to be analyzed on gel
Mix the resin with the rest of the reaction again by pipetting
After collecting samples for all time points, transfer the rest of the digestion mix to a fresh tube (should have ~60ul or 6ug left)
Purification of cleaved protein by Glutathione resin
GenScript Glutathione Sepharose 4B (cat # L00206, storage buffer 1x PBS with 20% ethanol)
1. Completely resuspend the Glutathione Resin by gently shaking the vial.
2. Transfer an appropriate amount of slurry to fresh tube. Usually 1 ml settled resin (2 ml 50% slurry) can bind 35-45mg GST-fusion protein (26 kDa).
3. Wash the Glutathione Resin with 10×bed volumes of cold (4°C) 1×PBS.
Aliquot 3x 5ul of Glutathione resin slurry
Wash resin with 50ul of cold 1xPBS (20x bed volume)
4. Add 50ul of STAT3 and PP7 digestion mix supernatant (after 20h) and 50ul of GST standard (@ 50ng/ul) to each tube containing
Add 5ul of pre-washed Glutathione resin
5. Incubate with gentle agitation (Thermocycler 600rpm) at RT for 30’
6. Spin the suspension @ 500xg for 5’ to sediment the gel
7. Transfer the supernatant to a fresh tube
8. Take 10ul of the supernatant for gel analysis
Add LDS and DTT to each sample (final 1x LDS, 50mM DTT)
Heat samples at 70C for 10’
Add 8ul of 1.1x LDS and 1ul of 0.5M DTT
Heat samples at 70C for 10’
Run 4-12% Bis-Tris gel
1) Marker
2) STAT3_0 (uncleaved)
3) STAT3_1h
4) STAT3_2h
5) STAT3_4h
6) STAT3_20h
7) STAT3_20h_after glut. resin
8) GST-PP7_0 (uncleaved)
9) GST-PP7_1h
10) GST-PP7_2h
11) GST-PP7_4h
12) GST-PP7_20h
13) GST-PP7_20h_after glut. resin
14) GST_500ng
15) GST_500ng after glut. Resin